There is known a neurotoxin including one produced by bacteria of Clostridium or by several fish and shellfish, typically a swellfish poison tetrodotoxin, a snake toxin alpha-bungarotoxin, and the like. These toxins, though different in their point of action, commonly block neurotransmission at the nerve end to thereby exhibit a muscular relaxation in mammals to which the toxins are inoculated. Among these, Clostridium toxin is a neurotoxin produced by bacteria of Clostridium which is divided into more than a hundred groups based on the form and function. For bacteria of Clostridium, Clostridium baratii, Clostridium butyricum, Clostridium botulinum, Clostridium tetani and the like are known. A botulinum toxin produced by Clostridium botulinum, aerobic Gram-positive bacteria, is the most lethal neurotoxin on earth. It is classified into seven serotypes, A, B, C, D, E, F and G, and the property of each serotype has been elucidated. The serotypes are distinguishable from each other by respective serotype-specific neutralizing antibodies. Depending on the serotypes, a botulinum toxin may vary in animal species it may affect, severity of paralysis it induces, duration of time of its action, and the like.
An active center protein of a botulinum toxin has a molecular weight of about 150 kDa (NTX) as common in all the known seven serotypes. Any botulinum toxin, when produced from Clostridium botulinum, is a complex composed of NTX and a relevant nontoxic protein. A serotype A botulinum toxin is produced in a molecular form of either 900 kDa (LL toxin), 500 kDa (L toxin), or 300 kDa (M toxin) These LL, L and M toxins are called a botulinum toxin complex. These botulinum toxins are, upon absorption in the upper small intestine, degraded to release a nontoxic protein and an active center protein, NTX, under alkaline conditions (in a lymphatic vessel). The released NTX is then bound to a receptor at the nerve end at its C-terminus of a heavy chain and taken into neurons via the receptor. Then, it specifically cleaves a protein in the presynaptic membrane through a light chain zinc methaloendopeptidase activity and inhibits a calcium-dependant release of acetylcholine to thereby block neurotransmission at the synapse (Non-patent reference 1).
Although a botulinum toxin is a neurotoxin that may lead human to death in botulinum intoxication through blockage of systemic neurotransmission, it may also be utilized as a remedy for treating a disease with an accelerated muscular tension such as e.g. dystonia by positively making use of its activity and by administering directly into the muscle of a patient suffering from the disease so that a local muscular tension may be relieved (Non-patent reference 2). For instance, a serotype A botulinum toxin complex (BOTOX; registered trademark) has been approved as a medicament for treating blepharospasm, strabismus, hemifacial spasm, and cervical dystonia, and for treating wrinkles at the middle of the forehead by the Food and Drug Administration (FDA). A type B botulinum toxin complex (MYOBLOC; registered trademark) has also been approved as a medicament for treating cervical dystonia by FDA. It is said that a serotype A botulinum toxin has a higher potency and a longer duration of action as compared to serotypes other than a serotype A botulinum toxin. An average duration of action of a serotype A botulinum toxin from its single muscular administration up till amelioration of symptoms is typically about 3 to 4 months.
Currently, a biological potential of a therapeutic preparation of a botulinum toxin such as a serotype A botulinum toxin is indicated as a mouse LD50 unit. One LD50 is defined as LD50 which is, based on intraperitoneal administration to mice, defined as an amount with which a half number of mice tested dies. Namely, a potential is quantified with a level or an amount of a neurotoxin with which mice die as a consequence of muscular relaxation. One LD50, i.e. one unit, in mice of commercially available serotype A botulinum toxin complex (Allergan, Inc., BOTOX; registered trademark; containing 100 units in a glass vial) is about 50 pg.
However, it is reported that assays for determining a potential of a serotype A botulinum toxin with LD50 unit in mice may vary widely from laboratory to laboratory (Non-patent reference 3). Some study planned for standardization of a serotype A botulinum toxin assay revealed that there was up to ten-fold difference in the results among 11 different laboratories (Non-patent reference 4). This variation however is not peculiar to an assay for a serotype A botulinum toxin. In fact, this assay has routinely been used as LD50 in a toxicity test for a number of chemical drugs, solvents, cosmetics and medical drugs but many administrative organizations gave up requiring the routine use of this LD50 for a toxicity test (Non-patent reference 5).
As such, as medical importance of the muscular relaxing activity of a botulinum toxin becomes highly recognized, an accurate quantification of the biological activity contained in a botulinum toxin preparation is needed in a manufacturing company and a laboratory as well as on clinical scene and up till the present a variety of quantification methods have been investigated.
Among the conventional methods for quantifying the botulinum toxin activity is a pinna reflex assay (Patent reference 1) where a botulinum toxin is administered to the levator auris longus muscle of rat and upon some duration of time quantification of the neurotoxin activity is performed with Electoromyograph using the auricle nerve. According to the teaching of this literature in which the auricle nerve is used and the cervical region of rat is excised for analysis, the same region needs be surgically excised for evaluating the effect of a botulinum toxin for a long period of time such as several days to several ten days. This however would be a burden to the animals used and thus it is not practical to use one and the same rat throughout the test but instead more rats will be necessary depending on days of the test. Furthermore, since a muscular region used for the test is small, said region is thought to be unsuitable for quantitatively evaluating a diffusion reaction in the muscle.
On the other hand, a method for determining the effect of a toxin (Patent reference 2), as reported, determines a potential of a botulinum toxin based on a muscular atrophy caused by administration of said toxin into the muscle of mammals. According to this method, however, a rat as administered with a botulinum toxin is sacrificed and the muscle at the site of administration is removed for analysis. Thus, for evaluating the effect of a botulinum toxin for a long period of time such as several days to several ten days, rats need be provided for respective days of measurement and hence many rats are necessary. Besides, as different rats are tested at each of days or under different conditions, a variation among the animals is thought to be high.
An electromyograph was also utilized for evaluating the effect of a botulinum toxin administered into the sternocleidomastoid muscle for therapeutic treatment of human suffering from cervical dystonia (Non-patent reference 5). For a surface electromyograph, a surface electrode is placed at a distance, usually of 1 to 3 cm, from the site of administration. A surface electrode may be utilized for measuring the magnitude and the range of a compound muscle action potential (CMAP) during the maximal voluntary contraction of the muscle to which the toxin is administered. It is envisaged that when a muscular paralytic effect initiated, a compound muscle action potential (CMAP) is decreased whereas, as a muscular paralytic effect is gradually waned, CMAP is increased. In this way, an electromyogram is used for judging the effect of a neurotoxin such as a botulinum toxin on the muscle or a group of muscles in individuals but non-quantitatively. The reason is that, as well known in the field of electric physiology, the electromyographic activity may vary among patients and, even in the same patient, may vary with the site of the muscle and days when administered. For instance, when the same patient is recorded simultaneously, the obtained repetitive surface electromyogram may significantly vary (i.e. from about 7% to about 20%). Furthermore, the range of the maximal voluntary contraction, as measured with surface electromyogram, may vary among patients.
In addition, the effect of administration of a serotype A botulinum toxin on a compound muscle action potential (CMAP) has been investigated using in vivo rat model (Non-patent reference 7). In this rat model, the effect on rat of difference in a dose of a neurotoxin is studied but quantification of a neurotoxin is not done.    Patent reference 1: U.S. Patent Publication No. 2003/0032891A1    Patent reference 2: Japanese patent publication No. 2005-509145 (WO2003/015829)    Non-patent reference 1: Jankovic, J. et al., Curr. Opin. Neurol., 1994, 7: p. 358-366    Non-patent reference 2: Ryuji Kaji et al., “Dystonia and botulinum therapy”, Shindan-To-Chiryosha, 2005    Non-patent reference 3: Schantz and Kautter, J. Ass. of Anal. Chem., 1978, 61: p. 96-99    Non-patent reference 4: Sesardic et al., Pharacol. Toxico. 1996, 78: p. 283-288    Non-patent reference 5: Pearce et al., Toxicol. App. Pharm., 1994, 128: p. 69-77    Non-patent reference 6: Dressler et al., Electromyographic quantification of the paralyzing effect of botulinum toxin in the stemocleidomastoid muscle, Eur. Neurol. 2000; 43: p. 13-16    Non-patent reference 7: Cichon, Jr., M D et al., Laryngoscope, 1995 Feb., 105(2): p. 144-148